Fe-S cluster containing proteins are ubiquitous and participate in diverse and essential cellular functions. The destruction of Fe-S clusters that occurs as a consequence of aerobic metabolism places a burden on cells (termed Fe-S stress) to replace essential proteins and deal with the resulting oxidative damage. Oxidative damage is an underlying cause of chronic and late onset diseases and therefore, it is important to decipher cellular responses to Fe-S stress. A long-term goal is to identify the pathways by which cells respond to Fe-S stress and avoid the potentially lethal oxidative damage caused by cluster destruction or loss of essential functions. This research plan studies Fe-S stress in the bacterium E. coli because there is a wealth of knowledge about the pathways of Fe-S cluster synthesis and the mechanisms of reactive oxygen species (ROS)-mediated Fe-S protein damage. The proposed studies will reveal the components of a Fe-S stress response that is directed by the Fe-S transcription factor, IscR in E. coli. The biochemical and cellular properties of [2Fe-2S]-IscR will be investigated to determine the role of oxygen and ROS in sensing Fe-S stress. These studies will test the model that Fe-S stress alters the function of IscR by changing the occupancy of the Fe-S cluster, thereby switching the suites of target genes in response to Fe-S occupancy of IscR. The DNA binding properties of IscR and the different classes of DNA sites recognized by this protein in the presence or absence of the Fe-S will be studied to understand how target genes recognition is regulated by Fe-S occupancy. The role of target genes in attenuating Fe-S stress will be investigated using genetic and molecular approaches. These studies will define how Fe-S proteins function as sensors of O2 or ROS and how they control transcription of target genes in response to Fe-S stress. The fundamental insights that we gain from the proposed studies will advance our understanding of Fe-S homeostatic mechanisms and the response to toxic ROS across biological systems. PUBLIC HEALTH RELEVANCE: Inadvertent reactions of iron and oxygen damage cellular components and over the long term lead to disease. We are studying the pathways that maintain a class of iron proteins at their appropriate levels for cell function and the mechanisms that attenuate cell damage when the pathways go awry.